CN103421697B - Aspergillus oryzae LJ366 strain used for degrading aureomycin - Google Patents
Aspergillus oryzae LJ366 strain used for degrading aureomycin Download PDFInfo
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Abstract
The invention relates to an Aspergillus oryzae LJ366 strain used for degrading aureomycin and belongs to the microbial technical field. The bacterial strain has already been preserved in the China General Microbiological Culture Collection Center on Aug 27, 2013 and the preservation number is CGMCC No.8078. The ITS gene sequence of the Aspergillus oryzae LJ366 strain used for degrading the aureomycin is composed of 593 bases. The bacterial strain can degrade the aureomycin and can be used for processing residual aureomycin in solid waste and liquid.
Description
Technical field
The present invention relates to a strain to degrade aspergillus oryzae (Aspergillus oryzae) LJ366 of duomycin, belong to microbial technology field.
Background technology
Duomycin (Chlortetracycline, CTC) belongs to tetracycline antibiotics, has growth promotion and low cost and other advantages under has a broad antifungal spectrum, lower concentration, is widely used in the industry such as livestock and poultry and aquaculture.After duomycin enters animal body, major part can not be absorbed by body completely, but excretes via ight soil with original shape or metabolite form, constitutes a threat to ecological environment security.Simultaneously, in Ferment of DM production process, a large amount of spoil and wastewater residual containing duomycin can be produced, if residual duomycin is dealt with improperly and enters environment, will have an impact to the quantity of non-targeted microorganisms and population structure, likely induce drug tolerant bacteria and resistant gene.At present, in water body and soil, resistant gene tetL, tetA, tetB, tetM, tetQ, tetO, tetW etc. that teracycline antibiotic residues causes have been detected.Therefore, the environmental pollution that the microbiotic such as duomycin cause has become one of hot issue of domestic and international common concern, the thorny problem also becoming duomycin manufacturing enterprise He utilize department urgently to be resolved hurrily.
Antibiotic remains in biological method degraded environment is utilized to be a kind of method with bright prospects.At present, in the screening of duomycin degradation bacteria strains, have and study report on a small quantity.2005, He Yu screened the strain obtained and can to degrade the monascus B3 bacterial strain of duomycin, can be used for process duomycin factory effluent.2012, the screening such as Li Yanju obtains 2 strains and can to degrade the bacterial strain of duomycin, be respectively trichoderma harziarum (Hypocrea lixii) LJ245 and penicillium spinulosum (Penicillium Spinulosum) LJ220, can be used for processing the waste water containing duomycin and solid waste.And the research of relevant aspergillus oryzae degraded duomycin aspect, there is not yet report both at home and abroad.
Summary of the invention
The object of the present invention is to provide a strain to degrade the bacterial strain LJ366 of duomycin and gene thereof.
Duomycin degradation bacteria strains LJ366 of the present invention, observe and 18S rDNA-ITS sequential analysis according to its morphological specificity, determine that this bacterial strain is aspergillus oryzae (Aspergillus oryzae), called after aspergillus oryzae LJ366(Aspergillus oryzae LJ366).This bacterial strain is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 27th, 2013, and preserving number is CGMCC NO.8078.Depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The ITS gene order of duomycin degradation bacteria strains LJ366 of the present invention is as follows:
Above-mentioned sequence is made up of 593 bases (bp).
Bacterial strain LJ366 grows comparatively fast on Ma Dingshi plate culture medium, the existing white flock of bacterium colony central authorities, mycelia is white flocculence, cultivate about 2d bacterium colony and produce color, initial stage is pistac, and along with growth fades to green and brown look, bacterium colony is often arranged in concentric wheel stripe shape, bacterium colony quality is thicker, and bacterium colony reverse side is milk yellow.Coated by spore on Ma Dingshi flat board, 28 DEG C-30 DEG C cultivations, about 2d spore germination, 4d colony diameter 3.0cm-3.5cm, 9d colony diameter 7.5cm-8.0cm, after 11d, bacterium colony is paved with whole flat board, and bacterium colony often has colourless transudate, in sparse droplet.Examine under a microscope, this bacterial strain mycelia is flourishing, transparent, has barrier film.Mycelia top produces top capsule, and the nearly flask shape of top capsule, conidium is spherical concatenating.
The bacterial strain LJ366 provided in the present invention can be sole carbon source with duomycin and grow in the concentration inorganic salt solid medium that is 0.01g/L-10.00g/L, is especially that in the inorganic salt plate culture medium of 0.01g/L-5.00g/L growth is better at chlortetracycline concentration.
By bacterial strain LJ366 of the present invention, be inoculated in and be sole carbon source with duomycin and chlortetracycline concentration is respectively in the inorganic salt liquid substratum of 0.5g/L and 1.5g/L, at 30 DEG C, 15d is cultivated in 170r/min concussion, and duomycin degradation rate can reach 91.59% and 80.98% respectively.
Bacterial strain LJ366 of the present invention has good Degradation to duomycin in bacterium slag, and to ferment 15d through bacterial strain LJ366, in non-sterilizing bacterium slag and sterilizing bacterium slag, the degradation rate of duomycin is reached for 90.56% and 98.11% respectively.
Bacterial strain LJ366 can tolerate strong acidic environment, is that in the duomycin bacterium slag of 2.2-2.5, this bacterial strain can better grow in initial pH value, by this bacterial strain metabolism, the pH value of bacterium slag can be made to rise to neutral to slight alkalinity.
Beneficial effect of the present invention:
Bacterial strain LJ366 has the function of degraded duomycin, can be sole carbon source with duomycin and grow in the substratum of concentration at 0.01g/L-10.00g/L.This bacterial strain is easy to use, to duomycin residual in liquid and solid waste, there is good Degradation, can be applicable to process duomycin residual in duomycin manufacturing enterprise waste and feces of livestock and poultry, meanwhile, be expected the biological restoration being applied to duomycin contaminate environment.
Accompanying drawing explanation
Fig. 1 is the Genomic DNA gel electrophorogram (1: genomic dna, M:Marker) of bacterial strain LJ366.
Fig. 2 be bacterial strain LJ366 PCR primer agarose gel electrophoresis figure (1:PCR amplified production, M:
Marker）。
Fig. 3 is the colonial morphology figure of bacterial strain LJ366.
Fig. 4 is the conidium fringe microgram of bacterial strain LJ366.
Fig. 5 is the ITS sequence phylogenetic tree of bacterial strain LJ366.
Fig. 6 is the degradation rate of bacterial strain LJ366 to the duomycin in shake-flask culture.
Embodiment
Embodiment 1
One, the selection systems of duomycin degradation bacteria strains LJ366
1, material prepares
Bacterium sample source: the duomycin bacterium slag of duomycin manufacturing enterprise.
Ma Dingshi liquid nutrient medium: KH
2pO
41.0g, MgSO
47H
2o0.5g, peptone 5g, glucose 10g, be dissolved in 1000mL tap water, 121 DEG C of moist heat sterilization 15min.Add agar in the ratio of 13%-18%, obtain Ma Dingshi solid medium.
Inorganic salt liquid substratum: (NH
4)
2sO
42.0g, K
2hPO
40.5g, KH
2pO
40.5g, MgSO
47H
2o0.5g, NaCl0.2g, CaCl
20.1g, FeSO
47H
2o0.01g, EDTA0.015g, be dissolved in 1000mL tap water, 121 DEG C of moist heat sterilization 20min.
In inorganic salt liquid substratum, add agar in the ratio of 13%-18%, obtain inorganic salt solid medium.
2, laboratory apparatus and equipment
Shimadzu LC-20A high performance liquid chromatograph, UNICO UV-2600A ultra-violet and visible spectrophotometer, Mettler Toledo ML204 analytical balance, Mettler Toledo pH meter, YT-CJ-IND Bechtop, HZQ-F160 full temperature shaking culture case, TG16-W supercentrifuge, YX-280D hand-held pressure steam oven, NiconEcolipase80i fluorescent microscope, DNA Engine PCR instrument, DcodeTM denaturing gradient gel electrophoresis instrument, DHP-9082 electro-heating standing-temperature cultivator, BOSCH refrigerator, Champ Gel-3200 gel imaging system etc.
3, bacterial strain screening
(1) isolation and purification of bacterial strain
Adopt plate streak analysis and purifying bacterial strain.Select duomycin bacterium slag many parts, with the bacterium slag that transfering loop picking is a small amount of, rule adding on the Ma Dingshi solid medium that chlortetracycline concentration is 1.0g/L, be placed in lucifuge under room temperature condition and cultivate 2-7d, obtain different bacterium colony.Then, provoke single bacterium colony, above-mentioned substratum continues line and carries out isolation and purification.This step repeats 3-5 time, until obtain single bacterium colony.
(2) screen
That step (1) separation and purification is obtained but single bacterium colony, be seeded to respectively in Ma Dingshi substratum and carry out activation culture, the inoculum size access of bacterial strain after activation press 3%-5% is sole carbon source with duomycin and in the chlortetracycline concentration inorganic salt liquid cultivation that is 0.5g/L, 30 DEG C, 170r/min light culture, at 3d and 6d, sample respectively, with efficient liquid-phase chromatography method, measure respectively and calculate the degradation rate of each bacterial strain to duomycin, obtaining the bacterial strain LJ366 that degradation rate is high.
Duomycin measuring method: get 1.0mL nutrient solution, with the membrane filtration of 22 μm, then measures duomycin content with high performance liquid chromatograph.HPLC condition: weighting agent is octadecylsilane chemically bonded silica, column temperature 35 DEG C, sample size is 10 μ L, determined wavelength 375nm, and moving phase is oxalic acid solution (0.01mol/L): acetonitrile: methyl alcohol volume ratio is 10:3:2, and flow velocity is 1.0mL/min.
Two, colony morphology characteristic is observed
Bacterial strain LJ366 grows comparatively fast on Ma Dingshi plate culture medium, the existing white flock of central authorities, mycelia is white flocculence, cultivate about 2d bacterium colony and produce color, initial stage is pistac, and along with growth fades to green and brown look, bacterium colony is often arranged in concentric wheel stripe shape, bacterium colony quality is thicker, and bacterium colony reverse side is milk yellow.Coated by spore on Ma Dingshi flat board, 28 DEG C-30 DEG C cultivations, about 2d spore germination, 4d colony diameter 3.0cm-3.5cm, 9d colony diameter 7.5cm-8.0cm, after 11d, bacterium colony is paved with whole flat board, and bacterium colony often has colourless transudate, in sparse droplet.Examine under a microscope, this bacterial strain mycelia is flourishing, and transparent, have barrier film, mycelia branch is many and irregular, overall as branch.Mycelia top produces top capsule, and the nearly flask shape of top capsule, conidium is spherical concatenating.Colonial morphology and the microscopic morphology of bacterial strain LJ366 are shown in Fig. 3 and Fig. 4 respectively.
Three, the molecular biology identification of bacterial strain LJ366
1, DNA extraction
The extraction of DNA comprises bacterial strain activation culture, cell wall breaking, DNA release, DNA abstraction and purification, the basic steps such as concentrated, the precipitation of DNA and washing.
(1) bacterial strain activation culture: preparation Ma Dingshi substratum, adopts 121 DEG C after packing, 15min autoclaving, inoculation LJ366 bacterial strain, 30 DEG C, 170r/min cultivates 2-3d.Then to be forwarded in new substratum under the same conditions, to continue to cultivate 2d.Activation culture 2-4 generation like this.
(2) the bacterium ball sterile filtration of step (1) being cultivated, the nutrient solution in bacterium ball is blotted with aseptic filter paper, the bacterium ball blotted is put into mortar liquid nitrogen fully grind, powder after grinding is proceeded in new 1.5mL EP pipe, add the SDS lysate of 600 μ L65 DEG C preheatings, 65 DEG C of insulation 30min, period constantly shakes up.
(3) in EP pipe, add equal-volume phenol/chloroform/primary isoamyl alcohol mixed solution (25:24:1) after being cooled to room temperature in ice, fully mix, 4 DEG C, 10000r/min, centrifugal 10min, get supernatant liquor in new EP pipe.
(4) in new EP pipe, add isopyknic chloroform/primary isoamyl alcohol mixed solution (24:1), fully mix, 4 DEG C, 10000r/min, centrifugal 10min, get supernatant liquor in new EP pipe.
(5) in supernatant, the dehydrated alcohol of 3M NaAc and the 2.5 times volume of 1/10 volume is added ,-20 DEG C of precipitation 30min.4 DEG C, 10000r/min, centrifugal 10min, abandons supernatant.
(6) 70% ice ethanol 500 μ L wash DNA2-3 time, and inversion is dried, and adds appropriate TE and dissolves.Preserve the DNA extracted for-20 DEG C.
(7) 1% agarose gel electrophoresis detect: get the DNA5 μ L in (6) step, add the loading buffer loading of 1 μ L.
(8) gel imaging system is observed and is taken a picture.Fig. 1 is the gel electrophoresis figure of bacterial strain LJ366 genomic dna.
2, pcr amplification
Adopt universal primer ITS1 and ITS4, pcr amplification is carried out to the ITS fragment of the 18S rDNA gene of bacterial strain LJ366.Pcr amplification sequence adopts 50 μ L reaction systems: 10XPCR Buffer5 μ L, primer I ST1 10mmol/L 1 μ L, primer I TS4 10mmol/L 1 μ L, template (about 25mmol/L) 1 μ L, rTaq archaeal dna polymerase (2U/ μ L) 0.5 μ L, dNTP (10 μMs) 1 μM, ddH2O 40.5 μ L.
PCR reaction conditions is: 94 DEG C of preheating 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, circulates 30 times, and 72 DEG C extend 10min.
3, PCR primer detects
Adopt the agarose gel electrophoresis method for detecting by 1% to detect PCR primer, and take a picture with gel imaging system, Fig. 2 is the rDNA-ITS fragment PCR products gel electrophoresis figure of bacterial strain LJ366.
4, order-checking and phylogenetic tree build
The ITS sequence of amplification is sent Beijing promise match genome research center order-checking, the 18S rDNA-ITS gene order obtaining bacterial strain LJ366 is as follows:
By sequencing result and ncbi database Blast comparison, the homology obtaining the ITS sequence of some bacterial strains in bacterial strain LJ366 and aspergillus oryzae (Aspergillus oryzae) and flavus (Aspergillus flavus) is 100%.Relevant Aspergillus strain sequence is searched in Gnenbank, sequence alignment is carried out with Clustalx software, analyze with MEGA5.05 software again, by adjacent method (Neighbor-Joining) phylogenetic tree construction (see figure 5), result shows, bacterial strain LJ366 and strains A spergillus oryzae NRRL35191 gathers in same branch, and Phylogenetic Relationships is the closest.
Four, bacterial strain LJ366 is accredited as a kind of New function bacterial strain
According to bacterial strain LJ366 colony morphology characteristic and molecular biology identification result, bacterial strain LJ366 is accredited as aspergillus oryzae (Aspergillus oryzae), and called after aspergillus oryzae LJ366(Aspergillus oryzae LJ366).This bacterial strain has been stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC), and preserving number is CGMCC NO.8078, and preservation date is on August 27th, 2013.Depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
At present, there is no the function that bibliographical information aspergillus oryzae (Aspergillus oryzae) has degraded duomycin both at home and abroad.Therefore, aspergillus oryzae LJ366 is the new strains that a strain has degraded duomycin function.
Embodiment 2
Bacterial strain LJ366 is growth and degradation experiment in sole carbon source substratum at duomycin
1, growth experiment on different concns mycin substratum
Preparation is sole carbon source with duomycin and chlortetracycline concentration is respectively the inorganic salt solid medium of 0.01g/L, 0.05g/L, 0.10g/L, 0.50g/L, 5.00g/L, 10.00g/L, 15.00g/L.Picking LJ366 bacterium colony is a little, and at the flat lining out of inorganic salt solid medium, under room temperature, lucifuge is cultivated.Learn through observing, at cultivation 3d, at chlortetracycline concentration be 0.01g/L, 0.05g/L, 0.1g/L, 0.5g/L and 5.0g/L flat board on grow mycelia, and well-grown.Cultivate 15d, at chlortetracycline concentration be 10.00g/L flat board on grow mycelia, illustrates bacterial strain LJ366 energy tolerable concentration be the duomycin of 10.00g/L.
2, bacterial strain LJ366 is to the degradation experiment of duomycin in shaking flask
Bacterial strain LJ366 is seeded to duomycin and is sole carbon source and chlortetracycline concentration is respectively in the inorganic salt liquid substratum of 0.5g/L and 1.5g/L, 30 DEG C, 170r/min shake-flask culture, sample respectively at 0d, 3d, 6d, 9d, 15d of cultivating, measure duomycin content in nutrient solution with high performance liquid chromatograph (HPLC).Duomycin measuring method is with embodiment 1.The degradation rate of duomycin is calculated according to measurement result.
Learnt by experimental result (see figure 6), cultivation 6d, bacterial strain LJ366 are that the degradation rate of the duomycin of 0.5g/L and 1.5g/L is respectively 56.37% and 47.55% to concentration; When cultivating 15d, its degradation rate can reach 91.59% and 80.98% respectively.
Embodiment 3
Bacterial strain LJ366 is to duomycin degradation experiment in bacterium slag
1, bacterial strain activation and the pre-treatment of bacterium slag
Bacterial strain LJ366 Ma Dingshi substratum activation culture.After bacterium slag crusher in crushing, be divided into two portions, a part adopts 121 DEG C of moist heat sterilization 20min-30min process, and another part is without wet heat treatment.
2, inoculation and fermentation culture
Respectively bacterial strain LJ366 after activation is inoculated into above-mentioned in pretreated bacterium slag respectively by the inoculum size of 5%-7%, and add a certain amount of carbon source respectively as wheat bran, sawdust or stalk etc., stir, put into fermenting container, under temperature is 30 DEG C of-35 DEG C of conditions, carry out solid fermentation, periodic agitation also samples.
3, in fermenting process, bacterium slag pH value measures
Take 4.00g bacterium slag respectively at 0d, 3d, 6d, 9d, 15d of fermentation, add 20ml distilled water, ultrasonic 30min, measure its pH value with pH meter.
Learnt by measurement result, bacterium slag pH value raises during the fermentation gradually, and at fermentation 6d, non-sterilizing bacterium slag pH value rises to about 6.8 by initial about 2.2, at fermentation 9d, rises to about 7.4, and afterwards, pH value maintains neutral to slight alkalinity.After sterilising treatment, the pH value of bacterium slag becomes about 2.5, and at the 9d of fermentation, sterilizing bacterium slag pH value rises to about 7.7.Illustrate that bacterial strain LJ366 better can grow in the bacterium slag that acidity is stronger thus.
4, the duomycin determination of residual amount in bacterium slag fermenting process
Bacterium slag 1.0000g is taken respectively at 0d, 3d, 6d, 9d, 15d of the fermentation of bacterium slag, add EDTA-Mcllvaine damping fluid 10.00mL, mixing, ultrasonic 30min, the centrifugal 3min of 1000r/min, get supernatant liquor, with 22 μm of membrane filtrations, utilize high performance liquid chromatograph to measure duomycin content in filtrate.Measuring method is with embodiment 1.According to measurement result, calculate duomycin degradation rate.
Learnt by experimental result, bacterial strain LJ366 has good Degradation to duomycin in bacterium slag, and to ferment 15d through bacterial strain LJ366, in non-sterilizing bacterium slag and sterilizing bacterium slag, the degradation rate of duomycin is respectively 90.56% and 98.11%.
EDTA-Mcllvaine buffer: take 28.41g Sodium phosphate dibasic, by water dissolution, is settled to 1000mL, is made into the disodium phosphate soln of 0.2mol/L; Take 21.01g citric acid, by water dissolution, be settled to 1000mL, be made into the citric acid solution of 0.1mol/L.The citric acid solution of 1000mL is mixed with 625mL disodium phosphate soln and is adjusted to pH=4, be made into Mcllvaine buffered soln.Take the Mcllvaine buffered soln that 60.5g disodium ethylene diamine tetraacetate puts into 1625mL, dissolve, shake up, for subsequent use.
Claims (3)
1. one strain degraded duomycin aspergillus oryzae LJ366, it is characterized in that: observed by morphological specificity, extracting genome DNA, 18S rDNA-ITS sequence amplification and comparison and phylogenetic tree construction, identify bacterial strain LJ366 be aspergillus oryzae (
aspergillus oryzae), called after aspergillus oryzae LJ366 (
aspergillus oryzaelJ366), this bacterial strain is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 27th, 2013, and preserving number is: CGMCC No.8078, and depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
2. the application of the aspergillus oryzae LJ366 of a strain degraded duomycin according to claim 1, is characterized in that: bacterial strain LJ366 is used in the degraded of duomycin.
3. the application of the aspergillus oryzae LJ366 of a strain degraded duomycin according to claim 2, is characterized in that: bacterial strain LJ366 has good Degradation to duomycin residual in liquid and solid waste; Bacterial strain LJ366 can be sole carbon source with duomycin and grow in the concentration inorganic salt solid medium that is 0.01g/L-10.00g/L; Bacterial strain LJ366 is seeded in and is sole carbon source with duomycin and in the chlortetracycline concentration inorganic salt liquid substratum that is 0.5g/L and 1.5g/L, 30 DEG C, 170r/min cultivates 15d, can reach 91.59% and 80.98% respectively to duomycin degradation rate.
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